A number of studies in recent years have clearly established that Drosophila S2 cells, which display properties similar to human macrophages, are an excellent system to study host-pathogen interactions. In particular, recent studies indicate that the intracellular pathogen Listeria monocytogenes can effectively enter and replicate in the cytosol of Drosophila S2 cells (Cheng and Portnoy, 2003; Mansfield et al. 2003). In order to identify host factors involved in Listeria infection, we have recently conducted a genome-wide RNAi screen for host factors involved in the infection process using Drosophila cells as a genetic system (Agaisse et al. 2005). We have identified more than 300 candidate genes involved in several aspects of the biology of Listeria infection, including cytoskeleton remodeling, vesicular trafficking, proteasome-mediated protein degradation and cell cycle. When individually depleted, these host factors lead to specific phenotypes corresponding to defects in specific stages of the infection, including entry, vacuole escape and intracellular growth. Here, we propose to conduct high-throughput screens to identify compounds affecting Listeria infection in Drosophila S2 cells. We have recently performed a pilot screen for compounds affecting Listeria infection using a library of 1,000 bioactive molecules and identified four potential candidates (see Preliminary Studies). We propose to conduct a similar approach on a large-scale format using a library of 20,000 compounds. We will test the specificity of the identified compounds by conducting secondary assays designed to assess cell viability, bacterial killing and pathogen specificity. We will then determine for a selected list of candidates which step of Listeria infection is affected by the activity of the compounds. We also propose to tentatively identify the targets of the compounds by conducting comparative analyses of the phenotypes observed in chemical and RNAi screens. Beyond the potential biomedical implications of the proposed chemical screen, we anticipate the identification of small molecules that will constitute new tools to explore the native cellular functions of the identified host factors.